Project Summary/Abstract Malaria has a devastating global impact on public health and welfare, with the majority of the world?s malaria cases occurring in sub-Saharan Africa. Malaria causes more than 400 thousand deaths every year, and about 3.4 billion people are at risk of contracting the disease. Anopheles mosquitoes are exclusive vectors of malaria, with species from the An. gambiae complex being the most important African vectors. Increasing insecticide resistance in anopheline mosquitoes now threatens the sustainability of current control efforts. A possible reason for the quick evolution and spread of insecticide resistance may be in the peculiar genetic architecture of malaria vectors. High levels of genetic diversity of Anopheles could mean that resistance mutations may already be present and that mosquito populations can quickly adapt to new insecticides. Therefore, population genomics studies uncover both challenges and opportunities for mosquito control in Africa. However, these analyses are limited by the availability of the single chromosome-level reference genome assembly for the An. gambiae PEST strain. In contrast, genomes of other major malaria vectors, An. arabiensis and An. coluzzii, are represented by numerous unmapped sequencing scaffolds; they have unidentified gaps and missing sequences, which may cause incorrect or incomplete annotation of genomic sequences; and they lack clear distinction between paralogous genes and genes from different haplotypes. As a result, these assemblies cannot be used as reliable references for population genomics studies and other chromosome-level analyses. Our long-term goal is to understand how natural genetic and chromosomal variations are connected with ecology and epidemiology of major malaria vectors. The central goal of this R21 is to develop, validate, and test high-quality genome assemblies for An. arabiensis and An. coluzzii. Toward this end, we propose three Specific Aims: Specific Aim 1. Develop genome assemblies for An. arabiensis and An. coluzzii using PacBio sequencing and chromosome-scale Hi-C scaffolding. Specific Aim 2. Validate the PacBio assemblies and construct physical genome maps for An. arabiensis and An. coluzzii. Specific Aim 3. Assess performance of the An. arabiensis and An. coluzzii genome assemblies as references for population genomics studies. Our expectation is that the integration of novel sequencing and scaffolding techniques with physical mapping will result in a more complete, chromosome-based assemblies that will facilitate studies of genetic diversity in An. arabiensis and An. coluzzii. Our hope is that by using species-specific genome assemblies, we may uncover important genomic variation that was missed when the An. gambiae assembly was used. This methodological advantage will remove an important limitation to uncover genetic determinants of their differing epidemiological contributions to disease transmission.